Research ArticleExperimental Studies
Open Access

Accessing Apoptosis Induction and Metastasis Inhibition Effect of Magnolol on Triple Negative Breast Cancer In Vitro

YU-CHING LI, CHUI-NA WONG, FEI-TING HSU, JIANN-HWA CHEN, CHUNG-CHI YANG, HENG-HAO LIU, WEI-LUNG CHEN and YUEH-SHAN WENG
In Vivo May 2023, 37 (3) 1028-1036; DOI: https://doi.org/10.21873/invivo.13177

Abstract

Background/Aim: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer that still requires improvement in treatment. Magnolol extract, derived from the bark of Magnolia officinalis, has traditionally been used in Asia to treat sleeping disorders and anxiety, and as an anti-inflammatory agent. Several reports have indicated that magnolol may have the potential to inhibit the progression of hepatocellular carcinoma and glioblastoma. However, the anti-tumor effect of magnolol on TNBC remains unknown. Materials and Methods: In this study, we used two TNBC cell lines, MDA-MB-231 and 4T1, to examine the cytotoxicity, apoptosis, and metastasis effects of magnolol. These were evaluated using MTT assay, flow cytometry, western blotting, and invasion/migration transwell assay, respectively. Results: Magnolol significantly induced cytotoxicity and extrinsic/intrinsic apoptosis in both TNBC cell lines. It also decreased metastasis and associated protein expression in a dose-dependent manner. Furthermore, the anti-tumor effect was associated with the inactivation of the epidermal growth factor receptor (EGFR)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT3) signaling pathway. Conclusion: Magnolol may not only induce cell death in TNBC through apoptosis signaling activation but also by down-regulating EGFR/JAK/STAT3 signaling, which mediates TNBC progression.

Key Words:

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer, which lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). High recurrence and metastasis rate limit therapeutic efficacy of standard treatments and result in poor outcome of TNBC patients (1, 2). Development of novel combination strategies is urgently needed to further enhance treatment response of TNBC.

Epidermal growth factor receptor (EGFR) is an upstream receptor kinase receptor that mediates tumor growth, survival, angiogenesis, invasion through activating downstream signaling pathways such as RAS/RAF/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK), phosphoinositide 3-kinases (PI3K)/AKT, and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways (3-5). Positive expression of EGFR is frequently observed and recognized as an effective therapeutic target in TNBC (6). EGFR inhibition was reported to evoke the suppression of TNBC but also sensitize TNBC cells to other therapeutic agents (6, 7).

Traditional Chinese medicine (TCM) is commonly accepted as an adjuvant therapy for treatment of breast cancer. TCM can reduce side effects induced by chemotherapy and radiotherapy in breast cancer patients (8-10). In addition, TCM was also found to improve invasive disease-free survival of TNBC patients (8). Herbal medicine is the most crucial part of TCM (11). Experiments in cell and animal models have shown that natural compounds found in medical plants mediate anti-TNBC mechanisms including induction of apoptosis and suppression of EGFR activation and its downstream pathways (12-14).

Magnolol is a multifunctional lignan compound isolated from the Chinese herbal plant Magnolia officinalis. It possesses anti-inflammatory, anti-oxidant, anti-diabetic, and neuroprotective capacity. In addition, several in vitro and in vivo studies showed that magnolol had a good safety profile while triggered tumor regression through blockade of growth factor signaling, initiation of apoptosis, and induction of cell cycle arrest (15-17). Liu et al. also found that magnolol effectively inhibited invasion ability of TNBC cells through inactivation of nuclear factor-kappaB (NF-Embedded ImageB) (18). However, the anti-TNBC properties of magnolol through apoptosis and the EGFR pathway have not yet been elucidated. Therefore, the main goal of present study was to evaluate mechanism of action of magnolol on apoptosis and EGFR signaling in TNBC cells.

Materials and Methods

Cell lines. The MDA-MB-231 human TNBC cell line and the 4T1 murine TNBC cell line were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Fremont, CA, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific). The cells were maintained in a 37°C incubator with 5% CO2 and 95% humidity.

Reagents and antibodies. The chemical reagents used in this study were purchased from Sigma (St. Louis, MO, USA) and were: 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), magnolol and dimethyl sulfoxide (DMSO). DiOC6 was purchased from Enzo Life Sciences (Farmingdale, New York, NY, USA). Antibodies against, MMP-9, Cyclin D1, XIAP, c-FLIP, UPA, VEGF-A, β-actin were obtained from Elabsicence (Houston, TX, USA). Antibodies recognizing MMP-2 (Proteintech; Rosemont, IL, USA), p-EGFR Tyr 1068, EGFR, p-STAT3 Tyr705, STAT3, p-JAK Tyr1034/1035 (Cell Signaling, Danvers, MA, USA) were purchased from the specified suppliers.

Cell viability (MTT assay). MDA-MB-231and 4T1 cells were seeded in 96-well plates, at a density of 5×103 cells/well overnight and treated with magnolol 0, 20, 40, 60, 80, and 100 μM for 48 h. The medium was replaced with 100 μl MTT reagent (0.5 mg/ml) for 2 h. Then, the MTT medium was replaced with 100 μl DMSO. The absorbance was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific) at 570 nm (19, 20).

Cleaved caspase-3, -8, -9 analysis. MDA-MB-231and 4T1 cells were plated in 6 well plates at a density of 2×105 cells/well, incubated overnight and treated with 0, 80, and 100 μM magnolol for 48 h. MDA-MB-231 and 4T1 cells were stained using cleaved caspase-3, caspase-8, and caspase-9 staining kit (CaspGLOW™ fluorescein staining kit, BioVision, Milpitas, CA, USA) incubate for 0.5 h at 37°C incubator following treatment. The activation of cleaved caspase-3, caspase-8, and caspase-9 after treatment were analyzed in the FL-1 channel using the NovoCyte flow cytometer with NovoExpress® system (Agilent Technologies Inc., Santa Clara, CA, USA) and the FlowJo (version 7.6.1) software for quantification (19, 21).

Fas and FasL analysis. MDA-MB-231and 4T1 cells were plated in 6 well plates at a density of 2×105 cells/well, incubated overnight and treated with 0, 80, and 100 μM magnolol for 48 h. After treatment, cells were trypsinized and stained with the FITC labeled anti-Fas (BioLegend) or the PE labeled anti-FasL (BioLegend) in 100 μl binding buffer and incubated for 40 min in the dark at room temperature. The Fas and FasL were detected in the FL-1 and FL-2 channel, respectively, using NovoCyte flow cytometry with NovoExpress® system and the FlowJo 7.6.1 software for quantification.

Mitochondria membrane potential (ΔΨm) analysis. MDA-MB-231and 4T1 cells were plated in 6 well plates at a density of 2×105 cells/well, incubated overnight and treated with 0, 80, and 100 μM magnolol for 48 h. Cells were trypsinized and stained with 1 μM DiOC6 in 500 μl PBS for 30 min at 37°C. The DiOC6 signal was detected in the FL-1 channel using NovoCyte flow cytometry with NovoExpress® software and the FlowJo 7.6.1 software for quantification.

Annexin-V/propidium iodide (PI) staining. MDA-MB-231and 4T1 cells were plated in 6 well plates at a density of 2×105 cells/well and incubated overnight. The FITC annexin-V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) was used for apoptosis detection on MDA-MB-231and 4T1 cells after treatment with 0, 80, and 100 μM magnolol for 48 h. After harvesting, cells were double stained with 1 μl annexin-V-FITC and 2 μl propidium iodide (PI) solution in 100 μl binding buffer for 15 min at room temperature. The Annexin V was determined in the FL-1 channel and PI was determined in the FL-2 channel using NovoCyte flow cytometry with NovoExpress® software (Agilent Technologies Inc.) and the FlowJo software (version 7.6.1) for quantification. The extent of early and late apoptosis were measured from the percentages of annexin-V (+)/PI (−) cells and annexin-V (+)/PI (+), respectively (22).

Invasion and migration assay. MDA-MB-231and 4T1 cells were plated in 10 cm plates at a density of 2×106 cells/well, incubated overnight and treated with 0, 80 and 100 μM magnolol for 24 h. The pretreated cells (2×105) were re-suspended in 200 μl serum-free medium and added to the upper transwell chambers (Corning, Corning, NY, USA) and allowed for migration and invasion. For the invasion assay, the transwells were pre-coated with matrigel one day before cell seeding. After 24 h of migration and 48 h of invasion, the transwells were fixed in 4% paraformaldehyde for 30 min at 4°C, stained with crystal violet solution for 2 h, photographed using microscope (Nikon ECLIPSE Ti-U, Minato, Tokyo, Japan), and staining was quantified by ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA) (20).

Western blotting assay. MDA-MB-231and 4T1 cells were plated in 10 cm dishes (2×106 cells) overnight and treated with 80 and 100 μM magnolol for 48 h. After treatment, cells were collected and total proteins were extracted using NP-40 lysis buffer containing proteinase inhibitor cocktail and phosphatase inhibitor (Sigma-Aldrich). The protein concentration was measured using the Bio-Rad Bradford method. In total, 50 μg protein extracts per group were separated in a 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA). Membranes were then blocked with blocking buffer (5% non-fat dry milk) and hybridized with primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence images were detected using the chemiluminescent image system MultiGel-21 (TOPBIO, New Taipei City, Taiwan, ROC) with TOPBIO Capture software (23).

Statistical analysis. Statistical significance was measured using one-way ANOVA with post hoc Tukey’s test; p-values p<0.05, p<0.01, p<0.001 were both considered statistically significant. Each value is displayed as mean±standard deviation (SD). The statistical analysis was conducted using the GraphPad Prism 7 (GraphPad, Boston, MA, USA). All experiments were repeated independently three times.

Results

Magnolol induced significant cytotoxicity and apoptosis in TNBC cells. Figure 1A shows the cytotoxic effect of magnolol on both MDA-MB-231 and 4T1 cells. The survival rate of both cell lines was reduced with increasing magnolol concentration and incubation time. MDA-MB-231 cells treated with 80 μM or 100 μM magnolol for 48 h showed a 50-60% reduction in viability compared to the control group. The viability of 4T1 cells also decreased to 50-60% after 24 h of treatment with 80 μM or 100 μM magnolol. We selected 80 and 100 μM magnolol, estimated to be the IC40 and IC50, respectively, for further experiments. To investigate whether magnolol-induced cytotoxicity is due to apoptosis induction in TNBC cells, we used annexin V/PI double staining, caspase-3 activation, and western blotting (Figure 1B-G). Figure 1B-D shows that magnolol significantly induced early and late apoptosis in TNBC cells. This effect was observed with increasing doses of magnolol. Additionally, magnolol treatment increased the expression level of cleaved caspase-3 (Figure 1E-F), while reducing the expression of anti-apoptotic proteins (C-FLIP, MCL-1, and XIAP) compared to the control group (Figure 1G). These results suggest that magnolol may induce cytotoxicity in TNBC cells through activation of the caspase-dependent apoptosis pathway.

Figure 1.
Figure 1.

The cytotoxicity and apoptosis effect of magnolol on triple-negative breast cancer. (A) MDA-MB-231 and 4T1 cells were treated with 0~100 μM magnolol for 24 and 48 h and assayed using MTT. (B) The expression of Annexin-V and (B-C) the quantification of early/late apoptosis following magnolol treatment of MDA-MB-231 and 4T1 cells were examined using flow cytometry. (E-F) The expression pattern of cleaved caspase-3 after magnolol treatment and its quantification are displayed. (G) The protein expression of MCL-1, c-FLIP, and XIAP after magnolol treatment examined using western blotting is shown. (a vs. 0 μM magnolol; b vs. 80 μM magnolol; 3p<0.005)

Magnolol induced the extrinsic and intrinsic apoptosis pathways in TNBC cells. To further elucidate the mechanism of magnolol-induced apoptosis, we first investigated whether death receptor-dependent apoptosis was affected. The results shown in Figure 2A-D indicate that magnolol enhanced the activation of Fas and FasL, ultimately resulting in the proteolytic cleavage of procaspase 8 (Figure 2E-F). In addition to the death receptor-dependent apoptosis (extrinsic apoptosis pathway), we also validated whether magnolol can trigger the intrinsic mitochondrial-dependent apoptosis pathway. As illustrated in Figure 2G-H, there was a higher loss of mitochondrial membrane potential (ΔΨm) after 48 h of treatment with 80 and 100 μM magnolol. Magnolol also stimulated the elevation of cleaved caspase-9 (Figure 2I-J). In summary, these findings suggest that the induction of apoptosis by magnolol is related to the activation of both extrinsic and intrinsic apoptosis pathways.

Figure 2.
Figure 2.
Figure 2.

Induction of the extrinsic and intrinsic apoptotic pathways by magnolol in triple-negative breast cancer. (A, C, E, G, I) The expression of Fas, Fas-L, cleaved caspase-8, DIOC6 and cleaved caspase-9 after magnolol treatment of MDA-MB-231 and 4T1 cells was examined using flow cytometry. (B, D, F, H, J) The quantification of the levels of the above-mentioned markers is displayed. (a vs. 0 μM magnolol; b vs. 80 μM magnolol; 2p<0.01; 3p<0.005)

Magnolol suppressed the invasion/migration effect and metastasis-related protein expression is associated with EGFR/JAK/STAT3 inactivation. To investigate whether magnolol may affect the tumor invasion and migration potential, we performed invasion and migration transwell assays. The area of invasion and migration of cells was markedly suppressed by magnolol treatment in both MDA-MB-231 and 4T1 cells (Figure 3A and B). We also examined whether cell invasion-associated genes in TNBC were downregulated by magnolol. As shown in Figure 3C, magnolol significantly reduced the levels of metastasis-associated proteins (MMP-9, MMP-2, UPA, and VEGF) and the proliferation-associated protein Cyclin D1. Moreover, we also investigated the effect of magnolol on EGFR, JAK and STAT3 activation using western blotting. The results indicated that the phosphorylation of EGFR, JAK and STAT3 was decreased by magnolol treatment in both MDA-MB-231 and 4T1 cells (Figure 3D). These results suggested that magnolol inhibited invasion/migration ability and expression metastasis-associated proteins in TNBC cells. The aforementioned effects may be associated with the inactivation of signal transduction mediated by EGFR, JAK, and STAT3.

Figure 3.
Figure 3.

The invasion/migration and expression of metastasis-related proteins in triple-negative breast cancer were inhibited by magnolol. (A) The invasion and migration patterns of MDA-MB-231 and 4T1 cells following treatment with magnolol are presented. (B) The quantification of the results using Image J is shown. (C) The protein expression of MMP-9, MMP-2, VEGF-A, uPA, and cyclin D1 following magnolol treatment, examined using western blotting is shown. (D) The protein expression of EGFR, JAK, and STAT3 after magnolol treatment examined using western blotting is presented. (a vs. 0 μM magnolol; b vs. 80 μM magnolol; 3p<0.005)

Discussion

Overexpression of metastasis-associated proteins such as MMP-9, MMP-2, VEGF-A, and uPA not only promotes tumor aggressiveness but it is also associated with poor survival of patients with breast cancer (24-26). Reduction of metastasis-associated proteins has been shown to inhibit migration, invasion, and angiogenesis in TNBC (27-29). Magnolol was showed to attenuate NF-Embedded ImageB-mediated MMP-9 expression and inhibit invasion of TNBC cells (18). Our results showed that magnolol inhibited expression of metastasis-associated proteins while effectively suppressed invasion and migration ability in MDA-MB-231 and 4T1 cells (Figure 3A-C). According to these data, it is suggested that downregulation of the above-mentioned metastasis-associated proteins is involved in magnolol-inhibited metastasis potential of TNBC cells.

Anti-apoptotic proteins such as MCL1, XIAP, and C-FLIP mediate tumor survival and associate with worse prognosis in patients with breast cancer (30-33). Suppression of anti-apoptotic proteins was found to not only trigger growth inhibition but also enhance sensitivity to therapeutic agents in TNBC. Both effective induction of apoptosis and inhibition of anti-apoptotic proteins contribute to tumor regression induced by therapeutic agents (31, 34). Magnolol was demonstrated to induce apoptosis through caspase-independent pathway in ER-positive breast cancer cells (35). Our results indicated that magnolol inhibited expression of anti-apoptotic proteins while significantly triggered apoptosis through extrinsic/intrinsic pathways in MDA-MB-231 and 4T1 cells (Figure 2). We also showed that caspase-3, -8, and -9 may participate in magnolol-induced apoptosis in TNBC cells.

JAK-STAT3, a critical oncogenic pathway, can be activated by EGFR signaling and activated STAT3 is a major driver promoting the malignant phenotype of cancer through upregulation of downstream effector molecules. Constitutive activation of STAT3 is conducive to expression of anti-apoptotic and invasion-associated proteins (36-38). The EGFR/JAK/STAT3 pathway is required for TNBC development and its disruption leads to suppression of tumor progression (38). Both overexpression of EGFR and p-STAT3 have been correlated with unfavorable survival outcome in TNBC patients (38, 39). Magnolol has been reported to attenuate EGFR and STAT3 activity in prostate cancer and glioblastoma cells (40, 41). Our data showed that protein levels of EGFR (Tyr1064), JAK (Tyr 1034/1035), and STAT3 (Tyr715) were reduced following treatment of MDA-MB-231 and 4T1 cells with magnolol (Figure 3D).

In conclusion, magnolol was shown to induce apoptosis through extrinsic and intrinsic pathways in TNBC cells. In addition, magnolol also alleviated EGFR/JAK/STAT3-mediated anti-apoptosis and invasion. We suggested that both effective induction of apoptosis and suppression of EGFR/JAK/STAT3 signaling pathway may be associated with magnolol-elicited suppression of TNBC cells.

Acknowledgements

The Authors thank the Medical Research Core Facilities Center, Office of Research & Development at China Medical University (Taichung, Taiwan, ROC) for the technical support.

Footnotes

  • Authors’ Contributions

    YCL, CNW, and FTH performed the experiments. JHC, CCY, HHL and YSW prepared the initial version of the article. YCL, CNW, FTH, WLC, and YSW designed the study, performed the literature review, and prepared the final versions of the article.

  • Funding

    This study was financially supported by a grant from Show Chwan Memorial Hospital, Changhua, Taiwan, R.O.C (grant number: BRD-110029) and Cathay General Hospital, Taipei, Taiwan, R.O.C. (grant number: CGH-MR-A11139). This work was also financially supported by the «Drug Development Center, China Medical University» from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest regarding the contents of this article.

  • Received March 5, 2023.
  • Revision received March 15, 2023.
  • Accepted March 16, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Accessing Apoptosis Induction and Metastasis Inhibition Effect of Magnolol on Triple Negative Breast Cancer In Vitro
YU-CHING LI, CHUI-NA WONG, FEI-TING HSU, JIANN-HWA CHEN, CHUNG-CHI YANG, HENG-HAO LIU, WEI-LUNG CHEN, YUEH-SHAN WENG
In Vivo May 2023, 37 (3) 1028-1036; DOI: 10.21873/invivo.13177
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